2, AppliChem GmbH, Ottoweg 4, Darmstadt, Germany
Today the rapid isolation of pure DNA samples is supported by a large number of commercial kits. These kits contain high affinity DNA-binding materials and all reagents for binding, purification, and elution. The two major DNA-binding materials integrated into these products are pure silica-matrices or silica-based anion-exchange resins [1,]. The very different physical characteristics of these two DNA-binding materials require different buffer systems and protocols for purification (see (Table 1) for a comparison).Table 1. Summary of characteristic features and differences between pure silica matrices and silica-based anion exchange resins.
Recently, maxXbond, the first regeneration system optimized for pure silica matrices has been developed [1,2,]. maxXbond allows a substantial cost reduction for DNA purification by the multiple usage of silica matrices.
Here we present maxXbond AX, the optimized regeneration kit for the multiple usage of silica-based anion exchange resins. The kit is based on the two regeneration buffers, RG1-AX and RG2-AX, and fulfills all requirements that have already been defined for maxXbond:
complete removal of all nucleic acids
no damage to the silica-based anion exchange resin
complete regeneration of the DNA binding capacity
The regeneration procedures for maxXbond AX have been adapted to the specific requirements of the larger and more complex matrices of silica-based anion exchange resins (see (Figure 1) A + B). First, RG1-AX is applied to the column. During the incubation time, all residual DNA molecules (either free or trapped in particles) are degraded and released from the column. Test incubations of up to 24 hours demonstrate that silica-based anion exchange resins are not damaged by RG1-AX. After RG1-AX has been removed from the column, RG2-AX is applied to remove and inactivate any residual RG1-AX and to regenerate the original DNA binding capacity.Figure 1A + B).
1A). For columns with low levels of DNA contaminations, RG1-AX is applied on top of the column. After the first milliliters have passed through the column, it is sealed and incubated for 60 minutes. This allows efficient contact of RG1-AX with the matrix. After this incubation, the tube is opened and RG1-AX is completely removed from the column. Finally, RG2-AX is applied to the column. After passage of RG2-AX through the column, it is ready for the binding of a new DNA sample.
1B). For columns with high amounts of contaminating DNA, the upside down procedure was developed. In this procedure, RG1-AX is applied by a syringe from the bottom of the column. During the 1 hour incubation, the buffer is passed through the column with the help of the syringe 3 to 5 times every 15 minutes. Then the buffer is removed by inversion of the column. Thereby all particles and contaminations on top of the membrane that covers the matrix are removed. To remove all traces of buffer RG1-AX the column is kept in the inverted position for one minute. Finally, RG2-AX is applied to the top of the column. After passage of RG2-AX through the column by gravity flow, it is ready for the binding of a new DNA sample.
There are two procedures for the application of maxXbond AX to the columns ((Figure 1) A + B). For the standard procedure RG1-AX and RG2-AX are pipetted on top of the columns and pass the matrix by gravity flow ((Figure 1) A). Care should be taken to allow a substantial incubation time with the solution RG1-AX for removal of all residual DNA molecules.
For thorough cleaning of heavily contaminated columns, we developed the upside down procedure that allows a much better and faster contact between RG1-AX and the matrix ((Figure 1)B). The additional advantage of this procedure is that all particles and contaminations trapped on the membrane that cover the top of the matrix are removed. The maxXbond AX-regenerated column can be used directly for a new DNA isolation cycle or can be stored indefinitely.
To verify that the maxXbond AX-regenerated columns have identical binding capacities and do not contain any traces of previous DNA samples, regenerated columns were used for the purification of a second DNA plasmid different from the first one. After elution and precipitation, the second DNA sample was dissolved in sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Analytical agarose gels [3,] and PCR analysis [4,] prove that no residual DNA from the first isolation is detectable in the second DNA plasmid sample (see (Figure 2)A + B).Fig. 2A + B).
2A). Four columns (1, 2, 3, 4) were used for 9 cycles of isolation of plasmid 1 always followed by regeneration with maxXbond AX. Finally, these columns were used for purification of plasmid 2. Identical aliquots of the eluted DNA samples from plasmid 1 and plasmid 2 were analyzed on an agarose gel. No traces of plasmid 1 are found in the preparation of plasmid 2 and all preparations give high amounts of DNA comparable to isolations from new columns.
2B). PCR analysis of plasmid 2 samples. Identical aliquots of 2 µl from the eluates of plasmid 2 were analyzed by analytical PCR. This test does not identify any DNA molecules from plasmid 1 from the previous isolation. Lanes: +P: Positive control using 50 ng plasmid 1 as template; -: Negative control without template DNA; +E: Control using 2 µl eluate from the regenerated column 1 and additional 25 ng plasmid 1 as template; 1, 2, 3, 4: PCR analysis using DNA preparations of plasmid 2 from regenerated columns 1, 2, 3, 4; M: Molecular weight marker.
Plasmid DNA isolated from maxXbond AX-regenerated columns fulfill all quality standards for molecular biology and is identical to plasmid DNA isolated from new columns. DNA preparations obtained from maxXbond AX-regenerated DNA binding columns were tested successfully in various molecular biology applications such as DNA plasmid screens, cloning, restriction digestions, isolation of DNA fragments, ligations, transformations etc.
Incubation of DNA samples from regenerated columns for 96 hours at 37°C did not show any degradation of the DNA, proving that DNA from regenerated columns are intact and have a high stability (data not shown).
The optimization of maxXbond AX for applications with silica-based anion exchange resins does not change the positive fundamental characteristics of maxXbond:
All components of maxXbond AX are biodegradable, harmless, or nontoxic
No aggressive acids or bases are used. No damage to material or equipment is observed even after prolonged incubation
Catalytic and cooperative properties of the solution components cause a rapid and efficient removal or degradation of biological molecules like membrane fragments, proteins, and nucleic acids
The new maxXbond AX regeneration system was tested with different commercially available DNA binding columns containing silica-based anion exchange matrices. The high-quality products from the leading suppliers gave no problems in 10 to 15 cycles of DNA binding, regeneration, and application of new samples. We noticed that some products from smaller companies did not show comparable quality standards and therefore these columns were not always suitable for the regeneration procedure.
In combination with the new refill system maxXmore AX the customers now have the opportunity to regenerate most of the commercially available silica-based anion exchange resins and to save substanial amounts of their DNA preparation costs.